HBeAg gene expression with baculovirus vector in silk worm cells.

Abstract

Miyanohara et al[1] first obtained products with the expression of HBeAg activity by constructing a yeast expression system; later researches discovered HBeAg expression in xeropus oocytes[2], COS cells[3], E. coli cells[4], Bacillus subtilis[5], which allow us to know more certainly about HBeAg gene. HBeAg has the same 149 amino acid sequence as the amino-terminal of HBcAg which is encoded by c-gene and consist s of 183 amid acids. The c-gene has a 89 bp pre-csequence in the upstream of it. The co-expressed product of the two genes (pre-c protein) is cleaved off the amino-terminal signal peptide sequence and the c-terminal alkaline region by hydrolases in the membrane of endoblasmic reticulum, forming the secretable HBeAg[6]. The most important thing is that the signal peptide encoded by pre-c region directs the formation and secretion of HBeAg, suggesting that only by eukaryotic expression systems can we produce HBeAg with high purity and activity. The domestic HBeAg/anti -HBe diagnostic kit is produced in E. coli cells, containing a high proportion of HBeAg which affects the quality of the kit. Recently, the technique with baculovirus vector to express foreign gene efficiently in worm cells and body has been applied and popularized[7]. We have replaced the polyhedron protein gene encoding sequence with human INF-α in Bombyx morinuclear poly-hedrosis virus( Bm NPV), suggesting that silk worm cells can recognize the signal peptide of human INF-α gene and cut correctly[8]. Ninety-nine percent protein becomes ma-ture only after the secreting stage, the Bm NPV-Bm N will be a good expression system. For this purpose, we amplified the pre-csignal peptide sequence and the same 149 amino acids sequence homologous with HBcAg at the N-end by PCR, and added appropriate restriction endonuclease sites on both 5’ and 3’ ends, cloned it into-Bm-NPV transfer vector pBmo30, the Bm-N cells were co-transfected by p-Bm-HBe and wild-type Bm-NPV DNA, and at length the recombinant virus with high expression HBeAg were efficiently obtainable after plaque purification.